1 Culture and identification of infectious agents, Lecture 25 Dr. Alvin Fox

2 Key Terms Isolation (culture) Agar plate plate/colonies Liquid media Identification & taxonomy Family Genus Species Type Strain After culture Biochemical (physiological) tests Genetic tests - Polymerase chain reaction (PCR) - Sequencing - DNA-DNA homology/arrays - Restriction enzymes (digests) Chemical - fatty acid/protein profiling Immunological Direct detection (i.e. without culture) PCR Antigen detection Staining (e.g. Gram stain) Serology (antibody detection)

3 Taxonomy Defines common traits among strains for a bacterial species Usually genetic Allows development of diagnostic kits

4 selecting discriminating features Species versus strains - selecting discriminating features

5 Classification Strain: one single isolate or line Strain: one single isolate or line Type: sub-set of species Type: sub-set of species Species: related strains Species: related strains Genus: related species Genus: related species Family: related genera Family: related genera

6 Streptococcus pyogenes Both terms define the species name Genus part Species part So Streptococcus pyogenes and Streptococus agalactiae would be two species in the same genus In a report On first usage: Streptococcus pyogenes On second usage: S. pyogenes

7 Identification of infectious agents in the diagnostic laboratory Aids treatmentAids treatment Helps antibiotic selectionHelps antibiotic selection General hospital laboratoryGeneral hospital laboratory –physiological tests More fully equipped laboratoriesMore fully equipped laboratories –Genetic tests

8 Steps in isolation and identification Step 1: Streaking culture plates Step 1: Streaking culture plates – colonies on incubation (e.g 24 hr) – size, texture, color, hemolysis – oxygen requirement

9 CDC/Dr. James Feeley Sheep blood agar plate culture Bacillus anthracisBacillus cereus.

10 Mixed colonies

11 Isolation and identification Step 2: Colonies Gram stainedStep 2: Colonies Gram stained – cells observed microscopically

12 Gram negative Gram positive Heat/Dry Crystal violet stain IodineFix Iodine Fix Safranin stain Alcoholdestain Alcohol de-stain

13 Gram stain morphology ShapeShape –cocci (round) –bacilli (rods) –spiral or curved (e.g. spirochetes) Single or multiple cellsSingle or multiple cells –clusters (e.g. staphylococci) –chains (e.g. streptococci) Gram positive or negativeGram positive or negative

14 Step 3: Isolated bacteria are speciated Generally using physiological testsGenerally using physiological tests

15 Clinical Microbiology Laboratory Bench

16 Step 4: Antibiotic susceptibility testing

17 No growth Antibiotic susceptibility testing Susceptible Not susceptible Bacterial lawn lawn Growth Antibiotic disk

18 Molecular differentiation GenomicsGenomics Gene characterizationGene characterization –PCR –Sequencing –Restriction digestion Hybridization (probes, arrays)Hybridization (probes, arrays) % guanine-cytosine% guanine-cytosine

19 16S rRNA Sequencing Differentiates bacterial speciesDifferentiates bacterial species Development of clinical tests based on sequence (e.g. PCR)Development of clinical tests based on sequence (e.g. PCR)

20 Real-time PCR Cycle one Cycle two Cycle ds DNA Dye

21 DNA-DNA hybridization 100% Homology + Heat 0% Homology Strain 1 Strain 2

22 Profiles Long chain fatty acids Long chain fatty acids - structural (e.g. cell membrane) Short chain Short chain - metabolic - volatiles - Fatty acids/alcohols

23 Protein profiling: defining a species by characteristic (low molecular weight) proteinsProtein profiling: defining a species by characteristic (low molecular weight) proteins Proteomics: defining all proteins expressed by a species under specific growth conditionsProteomics: defining all proteins expressed by a species under specific growth conditions

24 Bacterial DNA sequences can be amplified directly from human body fluids

25 Laboratory diagnosis without culture In general, when speed is of essence Of additional importance: The organism grows poorly/slowly The organism can’t be cultured

26 Microscopy spinal fluids (meningitis) spinal fluids (meningitis) sputum (tuberculosis) sputum (tuberculosis) sensitivity poor sensitivity poor

27 Streptococcal Agglutination Test Latex beads Streptococcal antigenic extract Antibody

28 Serologic identification antibody response to the infecting agent several weeks after an infection has occurred

Epidemics 29 associated with particular strains/types state or federal laboratory system